A variety of diseases are characterized by the intracellular accumulation of proteinaceous filaments. Abnormal protein aggregation in the form of fibrillar protein deposits (“fibrillization”), or amyloid plaques, characterizes many if not all neurodegenerative disorders, as well as degenerative diseases that affect the pancreas, heart, kidneys, and other tissues. For example, filaments comprised of the microtubule-associated protein tau accumulate within the neurons of patients afflicted with Alzheimer's disease, and filaments of α-synuclein form within affected neurons of basal ganglia in Parkinson's patients. Aggregation of the microtubule-associated protein tau into filamentous lesions is a hallmark pathology of Alzheimer's Disease (“AD”) and other tauopathic neurodegenerative diseases. There is increasing evidence that abnormal protein aggregation, both intra and extracellular, is a cause, rather than merely an effect, of these degenerative diseases. A great deal of circumstantial evidence has been reported in the scientific literature that suggests this causal influence. Genetic studies have linked genes encoding aggregated proteins to familial forms of a variety of diseases; animal modeling studies have shown that overexpression of aggregated proteins leads to disease associate phenotypes; and biophysical studies have shown that disease associated mutations to the genes encoding aggregated proteins promote protein aggregation in vitro.
Based on the evidence that abnormal formation of protein filaments influences the development of a variety of diseases, it would be desirable to design therapeutic strategies for interfering with the fibrillization process. One such therapeutic strategy is the targeted use of inhibitors of fibrillization. Identification and evaluation of the function of such inhibitors would be facilitated by an in vitro system in which filament formation can be reliably produced, controlled and observed. Optimally, such as system would permit the identification and evaluation of ihibitors of the discrete step or steps in the fibrillization process.
In vitro studies with one aggregating protein involved in AD, tau, have shown that fibrillization of full-length, unphosphorylated recombinant tau can be induced under near physiological conditions by treatment with either fatty acids, or the polyanionic substances RNA, and sulfated glycosaminoglycans, including heparin, dextran sulfate, and pentosan polysulfate. However, the time-frames for achieving fibrillization vary widely among these various inducers. For example, the timeframes for achieving fibrillization with polyanionic substances can be on the order of days, and are thus impractical for most high-throughput screening uses. Likewise, use of fatty acids such as arachadonic acid (“AA”) has disadvantages. The susceptibility of unsaturated fatty acids such as AA to oxidation, influences their activity and eventually renders them inert.
Accordingly, it would be desirable to have in vitro methods and model systems for identifying and evaluating potential inhibitors of protein fibrillization under physiologic conditions that are free of the limitations of existing methods. Such methods and model systems would not rely on labile reagents, and would be suitable for conducting rapid and high throughput in vitro screening assays of fibrillization inhibitors. Inhibitors identified using such methods and model systems would be useful for the treatment or prevention of disorders involving intracellular fibrillization, such as Alzheimer's and Parkinson's Diseases.